The recruitment of neutrophils out of the blood and into surrounding lung tissues is a critical event in pulmonary inflammation. For this to occur, neutrophils must first adhere to cell adhesion molecules (particularly E-selectin and ICAM-1) expressed on the surface of endothelial cells lining blood vessels. These adhesive interactions provide attachment and allow neutrophils to generate traction on the endothelial surface, so that they can migrate over it as they probe for endothelial junctions or other sites where they can cross the endothelial barrier. Engagement of these adhesion molecules also triggers signaling pathways in the endothelial cells that promote transmigration. Of the many signaling pathways that have been identified downstream from E-selectin and ICAM-1, several Rho family GTPases have been implicated in mediating the changes in the cytoskeleton and cell junctions that allow neutrophil passage. Little is known about the co-ordination of the different Rho proteins and how they become activated in response to E-selectin and ICAM-1 ligation. Additionally, it is not known whether tractional forces exerted by neutrophils on these adhesion molecules affect their signaling pathways to promote neutrophil transit across the endothelium. However, these processes are highly co- ordinated and tightly regulated to maximize the benefits of host defense and minimize the injury resulting from endothelial cell damage, particularly in the lungs where edema interferes with gas exchange. To tackle these questions, we propose the following specific aims. Aim 1 will examine the dynamics of activation of key Rho proteins (RhoA, Rac1, RhoG and Cdc42) in response to engagement and crosslinking of E-selectin and ICAM- 1 on lung microvascular endothelial cells. FRET-based biosensors for each Rho GTPase will be used to follow the time and location of their activation. Novel photomanipulation techniques will be used to activate or inhibit specific GTPases at precise times and places to examine how interactions of the GTPases affect neutrophil transmigration. Aim 2 will identify and manipulate guanine nucleotide exchange factors (GEFs) that are downstream of E-selectin and ICAM-1 and that regulate Rho protein activity. Aim 3 will determine whether tension on E-selectin and ICAM-1 initiates activation of Rho proteins. Using 3D force microscopy, we will examine whether mimicking the tension applied by neutrophils on E-selectin and ICAM-1 initiates or modulates signaling to Rho GTPases. Aim 4 will determine how neutrophil migration over endothelial cell surfaces induces tension along and across endothelial cells through E-selectin and ICAM-1, and whether their ligation modulates disassembly of VE-cadherin complexes. Taken together, the proposed studies address important signaling pathways that regulate neutrophil passage across the endothelium during inflammation. They will contribute to an integrated model of endothelial adhesion molecule signaling, incorporating spatial and temporal control that is novel and important to a comprehensive understanding of inflammation. These studies may identify new targets for therapies in the treatment of inflammatory diseases. PUBLIC HEALTH RELEVANCE: This grant seeks to understand how neutrophils (one type of white blood cell) adhere and pass through the endothelial cell lining of lung blood vessels in order to enter tissues during inflammation. Although inflammation and neutrophil recruitment is a normal aspect of host defense against pathogens and other injuries, the inappropriate recruitment of neutrophils underlies many inflammatory and immune diseases. Understanding the mechanisms underlying the steps in the process of neutrophil recruitment across an endothelial cell barrier may lead to the development of novel inhibitors and improved therapies.